Clostridium difficile associated disease (CDAD) is a major growing public health problem. The growth of C. difficile bacteria in the colon is thought to be inhibited by normal constituents of the mutualist bacteria flora. Therefore, the major trigger for the C. difficile infection is exposure to antibiotics which alter the composition of the normal intestinal microbiota. About 20% of patients with CDAD fail conventional therapy with antibiotics and develop recurrent disease. A significant fraction of these patients develop life-long dependency on antibiotics and often dysfunction of their gastrointestinal tract. Other patients develop fulminant disease, which is associated with high mortality rate. The protective role of the normal intestinal flora is supported by clinical success of bacteriotherapy by way of fecal transplantation. In this procedure the intestinal tracts of patients with recurrent CDAD are inoculated with fecal material from healthy donors. We had recently demonstrated in a case report that this procedure indeed is associated with establishment of donor bacteria in the recipient's colon. However, fecal transplantation is not widely available because of practical and aesthetic considerations. Our long-term goal is to develop a standardized, easy-to- administer formulation of colonic bacteria that can efficiently treat patients with CDAD. In this proposal we will use massively parallel pyrosequencing of hypervariable regions from SSU rRNA genes to characterize the composition of fecal material from patients with recurrent CDAD and their donors. In this exploratory work we will obtain critical data in pursuit of the following specific aims: 1) Determine the composition and diversity of the patient fecal microbiome before and after bacteriotherapy, 2) Determine the rate of recolonization and stability of the fecal microbiome following bacteriotherapy, and 3) Determine if a fecal sample from a single donor can be preserved and later used to restore normal bowel functioning to several CDAD patients. The data obtained from the first aim will form the foundation for future extension of this work requiring more patients. The results will allow us to perform power calculations to determine the number of patients needed in such studies to obtain more definitive results. Bacteriotherapy of patients with CDAD represents a unique opportunity to study establishment of new microflora in an adult patient. The data obtained from the second aim will lay the foundation into future studies that will be further investigate host- microbial interactions in this unique clinical situation. Finally, the third aim represents the first step toward our ultimate goal and has the potential to significantly reduce the complexity of the pursuit for the signature of healthy, protective colonic microflora. PUBLIC HEALTH RELEVANCE: Clostridium difficile is the major known cause of antibiotic associated disease, and represents a growing problem. We are normally protected against this infection by normal bacteria that live in our intestines. Antibiotics used in medical practice weaken this protection. A significant fraction of patients with this infection cannot be treated by conventional treatments, which also involve antibiotics. Our ultimate goal is to develop a standardized formulation of protective bacteria. In this grant we will make critical initial steps toward this goal by studying composition of intestinal bacteria in patients undergoing fecal transplantation for C. difficile infection refractory to conventional treatment.